The entry of sulfate into metabolism requires that it be chemically activated. The only known metabolic means of activating sulfate is the formation of the very high-energy phosphoric-sulfuric acid anhydride bond (?Go= -19 kcal/mole). This bond is the chemical hallmark of activated sulfate (APS or PAPS), and it is from this high-energy environment that the sulfuryl-moiety (-SO3) passes quickly and favorably into its subsequent metabolic biochemistry. The activated bond is formed in a transfer reaction, catalyzed by ATP sulfurylase, in which the adenylyl-moiety (AMP~) of ATP is transferred to sulfate. In mammals, sulfuryl-group transfer to proteins and small molecule metabolites regulates a wide-variety of metabolic processes including neuropeptide- and steroid-hormone action, growth-factor recognition, and lymph cell circulation. This proposal outlines structurally-based mechanistic inquires designed to address central issues regarding the function and evolution of the mammalian class of ATP sulfurylases. Bacterial ATP sulfurylases harbor a GTPase subunit (discovered in this laboratory) that is an evolutionary descendant of elongation factor Tu. The conformational changes that this subunit undergoes as a consequence of GTP hydrolysis accelerate turnover of the adenylyl-transferase subunit, and couple the chemical potentials of GTP hydrolysis and APS synthesis. We have recently discovered that ATP sulfurylase forms a complex with another enzyme in the cysteine biosynthetic pathway (O-acetly-l-serine sulfhydrylase), and that their interactions produce "new" catalytic function - the hydrolysis of ATP. These enzymes organize into a metabolic pump, each stroke of which delivers one molecule of APS into the pathway. The mechanism of the pump will be explored in this grant. Working with an as yet uncharacterized and novel ATP sulfurylase from Mycobacterium tuberculosis, our preliminary data extends these finding to include five of the seven enzymes in the pathway. We will define and study the cysteine metabolon with the goal of understanding the hierarchical functions that emerge from the self-organization of this pathway.